A canine coronavirus attenuated strain CCoV AS22 and its application

Abstract

The present invention discloses a canine coronavirus attenuated strain CCoVAS22 and its application, and relates to the field of biomedicine technology. The canine coronavirus attenuated strain CCoVAS22 was deposited in the China Center for Type Culture Collection on July 12, 2024, and the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: V202413. The present invention utilizes the technical means of cross-passaging in MDCK and CRFK cell lines to obtain an independently isolated CCoVAS22 strain, which has the advantages of high toxicity and stable passage on cells. After inactivation by β-propiolactone, it has the characteristics of high safety and good immunogenicity. It can be used as a candidate strain for CCoV inactivated vaccine, which can alleviate the problem of the small number of canine coronavirus isolates in China to a certain extent, and provide technical support for the prevention of diseases caused by canine coronavirus.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a canine coronavirus attenuated strain CCoV AS22 and an application thereof. Background Art

[0002] Canine coronavirus disease (CCoV) is caused by canine coronavirus (CCoV) and primarily infects canines such as dogs, raccoon dogs, and foxes. The highest incidence occurs in puppies aged 2-4 months. When infected alone, CCoV typically causes self-limited enteritis and mild diarrhea in dogs. However, co-infection with other pathogens can lead to severe clinical symptoms and even death.

[0003] Research on CCoV in China started later than abroad, and effective methods for isolating the pathogen are still immature, resulting in a limited number of isolates obtained in my country, which has restricted CCoV research. Currently, there are no specific drugs for treating CCoV viral enteritis, and prevention of the disease relies primarily on vaccination. With the recent reports of multiple fatal cases of highly pathogenic CCoV variants in Europe, increasing research interest in CCoV vaccines has been growing. This present invention proposes to develop a canine coronavirus attenuated strain, thereby providing technical support for preventing diseases caused by canine coronavirus. Summary of the Invention

[0004] The present invention aims to provide a canine coronavirus attenuated strain, CCoV AS22, and its use to address the aforementioned problems of the prior art. This inactivated strain exhibits high safety and good immunogenicity. Vaccines prepared with this strain can protect against infection with some prevalent CCoV strains in China, providing technical support for the prevention of diseases caused by canine coronavirus.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a canine coronavirus (Canine coronavirus), which was deposited in the China Center for Type Culture Collection on July 12, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCCNO: V202413.

[0007] The present invention also provides use of the attenuated canine coronavirus strain CCoV AS22 in preparing a drug for preventing diseases caused by canine coronavirus.

[0008] Furthermore, the drug is a vaccine.

[0009] Furthermore, the vaccine is an inactivated vaccine.

[0010] The present invention also provides a medicine for preventing diseases caused by canine coronavirus, wherein the active ingredient includes the above-mentioned canine coronavirus attenuated strain CCoV AS22.

[0011] Furthermore, the drug is a vaccine.

[0012] Furthermore, the vaccine is an inactivated vaccine.

[0013] Furthermore, the vaccine also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the auxiliary material includes aluminum hydroxide adjuvant.

[0015] The present invention also provides a method for preparing an inactivated canine coronavirus vaccine, comprising the following steps: inactivating the attenuated canine coronavirus strain CCoV AS22 to obtain an inactivated virus antigen;

[0016] The inactivated virus antigen and the vaccine adjuvant are evenly mixed to obtain the canine coronavirus inactivated vaccine.

[0017] The present invention discloses the following technical effects:

[0018] The present invention utilizes the technical means of cross-passaging in MDCK and CRFK cell lines to obtain an independently isolated CCoVAS22 strain. The strain has the advantages of high toxicity and stable passage on cells. After inactivation by β-propiolactone, it has the characteristics of high safety and good immunogenicity. It can be used as a candidate strain for CCoV inactivated vaccine, which can alleviate the problem of the small number of canine coronavirus isolates in China to a certain extent, and provide technical support for the prevention of diseases caused by canine coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 This is the growth curve of CRFK cells infected with CCoV AS22;

[0021] Figure 2The figure shows the electrophoresis diagram of RT-PCR detection of exogenous virus of CCoV AS22 virus. From left to right, lane 1: 2000bp DNA marker; lane 2: CAV positive control (1030bp); lane 3: CCoV positive control (409bp); lane 4: CDV positive control (455bp); lane 5: CPV positive control (559bp); lane 6: CPIV positive control (182bp); lane 7: CAV negative control (no band); lane 8: CCoV negative control (no band); lane 9: CDV negative control (no band); lane 10: CPV negative control (no band); lane 11: CPIV negative control (no band); lane 12: CCoV AS22 virus sample (CAV negative, no band); lane 13: CCoV AS22 virus sample (CCoV positive 409bp); Lane 14: CCoV AS22 virus sample (CDV negative, no band); Lane 15: CCoV AS22 virus sample (CPV negative, no band); Lane 16: CCoV AS22 virus sample (CPIV negative, no band);

[0022] Figure 3 The results of the exogenous virus test using IFA on the 10th generation strain of CCoV AS22;

[0023] Figure 4 TCID for long-term storage of CCoV AS22 strains at different temperatures 50 Toxicological price test results;

[0024] Figure 5 These are some microscopic pathological changes in the tissues of dogs after inoculation with the CCoV AS22 strain. A to D are HE-stained tissue sections of the spleen (A), jejunum (B), ileum (C), and cecum (D) of the inoculated dogs, respectively.

[0025] Figure 6 This is the result of neutralizing antibody test after dogs were immunized with CCoV AS22 inactivated vaccine. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1

[0032] 1. Isolation and culture of CCoV AS22 strain

[0033] 1.1 Cells and viruses

[0034] CRFK and MDCK cells were provided by Wuhan Keqian Biotechnology Co., Ltd. These cells were cultured in 10% FBS-DMEM medium at 37°C in a 5% CO2 incubator. The CCoV AS22 strain was isolated, identified, and preserved from a canine anal swab by the Rabies Virus Laboratory of Veterinary Public Health at Huazhong Agricultural University.

[0035] 1.2 Virus culture and passage

[0036] 1.2.1 Virus culture

[0037] The CCoV AS22 strain was inoculated into the well-grown cell monolayer, adsorbed for 1 h, the inoculum was discarded, and 1% FBS-DMEM medium was added to continue culturing in a 37°C, 5% CO2 incubator. After 72 h, when more than 80% of the cells became pathological, the culture medium was frozen and thawed once at -20°C to -80°C, and the cell culture virus liquid was harvested.

[0038] 1.2.2 Cell passaging method:

[0039] The MDCK / CRFK cell cross-passaging method was used. The CCoV AS22 virus was first cultured in MDCK cells (inoculated into new MDCK cells at a ratio of 1 / 10), serially passaged to the fifth generation, and then passaged to the 60th generation on CRFK cells using the same method.

[0040] 1.3 Virus growth curve and TCID 50 Determination

[0041] CCoV AS22 was passaged to the sixth generation, and the virus solution was appropriately diluted and inoculated into CRFK cells cultured in 12-well plates at MOIs of 0.01, 0.05, 0.1, and 1, respectively. Three replicate wells were used, and one 12-well plate was taken out every 12 hours and temporarily stored at -80°C until 72 hourspi. After freezing and thawing once, TCID 50 Determination.

[0042] CCoV virus culture was serially diluted 10-fold with maintenance solution, 10 -1 ~10 -10 CRFK cells were inoculated into 96-well plates at 100 μL / well, with 8 replicates, and cultured in a 37°C, 5% CO2 incubator until 72 hpi. Cytopathic effects were observed under a microscope, and TCID was calculated according to the Reed-Muench method. 50 The results are shown in Figure 1 , CCoV AS22 with an MOI of 0.05 had the highest titer 48 hours after infection, and the highest titer was 10 5.5 TCID 50 / 0.1mL.

[0043] 1.4 Mycoplasma testing

[0044] Tested according to the current appendix of the Chinese Veterinary Pharmacopoeia, no mycoplasma growth was found.

[0045] 1.5 Exogenous virus testing

[0046] 1.5.1 The product must be tested according to the appendix of the current Chinese Pharmacopoeia of Veterinary Medicine and free from exogenous viral contamination.

[0047] 1.5.2 PCR test for exogenous viruses

[0048] Using CCoV AS22 10th generation virus as template, PCR amplification was performed with specific primers for canine adenovirus (CAV1 / CAV2) E3 protein gene and canine parvovirus (CPV) VP2 gene; RT-PCR amplification was performed with specific primers for CCoV M protein gene, canine distemper virus (CDV) H gene, and canine parainfluenza virus (CPIV) N gene. The primer sequences are shown in Table 1. The results showed that the CCoV AS22 10th generation virus sample was positive only for the CCoV M protein gene, while the amplification results of other exogenous virus primers were negative. Figure 2 At the same time, IFA was used to test the exogenous virus of CCoV AS22 10th generation virus, and the results are shown in Figure 3 , further proving that the CCoV AS22 virus strain is not contaminated by related exogenous viruses.

[0049] Table 1 Primers used in PCR testing of exogenous viruses

[0050]

[0051] 1.6 Microbial Deposit

[0052] The present invention submits the CCoV AS22 strain to a preservation institution for preservation, and its microbial preservation number is CCTCC NO: V202413; the preservation time is: July 12, 2024; the preservation unit is: China Center for Type Culture Collection; the preservation address is: Wuhan University, Wuhan, China.

[0053] 2. CCoV AS22 virus seed shelf life test

[0054] CRFK cells were cultured to prepare CCoV AS22 virus, and the virus was aliquoted into 1 mL / vial. The aliquots were stored at -80°C, -20°C, and 4°C respectively. At regular intervals, one aliquot was taken out from each aliquot to determine the TCID of the virus. 50 , evaluate the shelf life of CCoV virus strains. Figure 4 The virus was stored in -80℃ freezer for 12 months. 50 No significant change, stored in -20℃ refrigerator for 12 months TCID 50 1 titer drop; stored at 4℃ for 3 months TCID 50 The results showed that the virus seed should be stored at -80℃ for at least 12 months.

[0055] 3. CCoV AS22 virulence test

[0056] 3.1 Experimental dogs

[0057] Eight dogs aged 7-8 weeks without maternal antibodies (serum CCoV neutralizing antibody titer ≤ 1:2) were provided by the experimental animal base (breeding dogs were not vaccinated). The experimental dogs were housed in isolation in the animal room of Huazhong Agricultural University and fed a commercial diet with free access to water. After acclimation for one week, they were used in this study. Starting 3 days before the experiment (day -3), fecal samples were tested using a CCoV rapid antigen detection kit (Anjie) (IC) to confirm the absence of CCoV infection.

[0058] 3.2 Grouping and virus inoculation

[0059] Six dogs aged 7-8 weeks were selected, and four of them were vaccinated with 10 4 TCID 50 1 mL of CCoV AS22 virus was administered intranasally or orally. Two other dogs were inoculated with the same volume of cell maintenance medium as a control group.

[0060] All dogs except dog #4 were observed for 14 days after virus inoculation. Clinical symptoms of the experimental dogs were observed daily, rectal temperature was monitored, fecal toxin excretion was detected using immunochromatographic test strips (IC) and virus isolation (VI) methods, and anticoagulated blood was collected every 3 days for white blood cell differential count.

[0061] On the 7th day after virus inoculation, dog No. 4 in the experimental group and dog No. 6 in the control group were anesthetized, killed, and dissected to obtain relevant tissue samples. After formaldehyde fixation, sectioning, and HE staining, pathological histological observation was performed under a microscope.

[0062] 3.3 Virulence test results of virus strains

[0063] The results of the virulence test of CCoV AS22 are shown in Table 2. During the entire observation period, the body temperature of dogs inoculated with CCoV AS22 (1#, 2#, 3#, 4#) occasionally increased, and obvious clinical symptoms appeared after vaccination, mainly manifested as depression, decreased food intake, and diarrhea. The lymphocyte count increased acutely 3 days after vaccination, and in severe cases it increased to 43.7%. The dogs showed obvious inflammatory reactions. The results of histopathological observation of dog 4# showed that pathological changes mainly occurred in the spleen, jejunum, ileum, and cecum, with splenic lymphocyte reduction and bleeding. The villi of the jejunum partially fell off, the goblet cells decreased, and lymphocytes and plasma cells were scattered. The epithelium of the villi of the ileum fell off, the goblet cells decreased, and lymphocytes and plasma cells were scattered. The villi of the cecum partially fell off, lymphocytes and plasma cells were scattered, the intestinal glands were dilated, and necrotic cell fragments were occasionally seen in the intestinal gland cavity. There were no obvious pathological changes in other tissues. Figure 5 The healthy control dogs (5# and 6#) remained normal throughout the observation period, and no pathological changes were observed in the histopathological observation of dog 6#.

[0064] Table 2 Virulence and safety characteristics of CCoV AS22 strain

[0065]

[0066] Dpi: days after virus inoculation; IC: colloidal gold test strip detection; VI: virus isolation detection; A: depression, loss of appetite; B: diarrhea; “none”: no clinical symptoms; “+” indicates positive, “-” indicates negative.

[0067] 4. Inactivated CCoV vaccine

[0068] The inactivated canine coronavirus vaccine was prepared using the CCoV AS22 strain.

[0069] 4.1 The brief process of virus culture and inactivation is as follows: CCoV AS22 is cultured in CRFK cells, centrifuged at 4000 rpm to remove cell debris, and the supernatant is inactivated by adding 1‰ of β-propiolactone to obtain CCoV AS22 inactivated virus antigen, which is stored at 4°C until use.

[0070] 4.2 Preparation of CCoV inactivated vaccine with aluminum hydroxide adjuvant

[0071] Take the above CCoV AS22 inactivated virus antigen, slowly add an equal amount (V:V) of commercial aluminum hydroxide gel adjuvant (adjuvant 2%, InvivoGen USA), mix well, and make a final virus particle content of 10 6 TCID 50 inactivated vaccine.

[0072] The amino acid sequence of the target antigen protein, Spike protein, designed for this inactivated vaccine (SEQ ID NO. 1) is as follows:

[0073]

[0074] 4.3 Immunization

[0075] Eight dogs aged 5-6 weeks (serum CCoV neutralizing antibody titer ≤ 1:2) were randomly selected from five dogs as the experimental group and inoculated with 1 mL / dog of the inactivated vaccine prepared in 4.2. Three other dogs were similarly inoculated with an equal volume of cell maintenance medium as the control group. Both the experimental and control groups were vaccinated subcutaneously, at weeks 0 and 3. The second vaccination dose and route were identical to the first. The dogs were observed for an additional two weeks, with blood samples collected weekly for neutralizing antibody determination.

[0076] 4.4CCoV serum neutralizing antibody assay

[0077] Canine serum was inactivated at 56°C for 30 min before testing. Serum was serially diluted in 2% FBS-DMEM medium at a ratio of 1:2 (50 μL system) in a 96-well cell culture plate. Each dilution was repeated in 4 rows of wells, and 50 μL / well containing 200 TCID 50 The CCoVAS22 virus solution was incubated at 37°C for 1.5 hours, and 100 μL / well (about 60% cells) of the 96-well CRFK cell plate prepared the day before was added. The cells were cultured in a 37°C CO2 incubator for 4 days. The CPE was observed under a microscope, and the neutralizing antibody titer was calculated based on the logarithm of the maximum dilution of serum that completely inhibited the cytopathic effect. The results are shown in the table. Figure 6 The neutralizing antibody titer of the secondary immunization with the CCoV AS22 inactivated vaccine reached 1:181, indicating good immunogenicity.

[0078] In summary, the CCoV AS22 strain has potential application value in developing into an inactivated vaccine.

[0079] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A canine coronavirus (CCoV) attenuated strain CCoV AS22, characterized by: The canine coronavirus attenuated strain CCoV AS22 was deposited in the China Center for Type Culture Collection on July 12, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: V202413.

2. Use of the attenuated canine coronavirus strain CCoV AS22 as claimed in claim 1 in the preparation of a medicament for preventing diseases caused by canine coronavirus.

3. The use according to claim 2, characterized in that The medicine is a vaccine.

4. The use according to claim 3, characterized in that The vaccine is an inactivated vaccine.

5. A drug for preventing diseases caused by canine coronavirus, characterized in that: The active ingredient comprises the canine coronavirus attenuated strain CCoV AS22 according to claim 1.

6. The drug according to claim 5, characterized in that The medicine is a vaccine.

7. The drug according to claim 6, characterized in that The vaccine is an inactivated vaccine.

8. The drug according to claim 6, characterized in that The vaccine also includes pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that The auxiliary material includes aluminum hydroxide adjuvant.

10. A method for preparing an inactivated canine coronavirus vaccine, characterized in that: The method comprises the following steps: inactivating the canine coronavirus attenuated strain CCoV AS22 according to claim 1 to obtain an inactivated virus antigen; The inactivated virus antigen and the vaccine adjuvant are evenly mixed to obtain the canine coronavirus inactivated vaccine.

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